Study On The Epigenetic Mechanism Of Hepatitis B Virus X Protein In Hepatocellular Carcinoma

Liver cancer is the fifth most common cancer in the world and the third most commoncause of cancer-related death; it is a serious threat to human health. And the most importantrisk factor for liver cancer is hepatitis virus infection. Overall,50%to55%of cases ofprimary liver cancer are attributable to persistent hepatitis B virus (HBV) infections. HBVcauses chronic infection in approximately400million people in the world. It is estimatedthat50%of male carriers and14%of female carriers will eventually die of thecomplications of cirrhosis and hepatocellular carcinoma (HCC). But the pathogenesis ofliver cancer is not very clear at present. Classic genetics has provided a firm foundation forour understanding of carcinogenesis, accumulation of genetic changes during tumorprogression has been well documented for many tumors, and numerous genes that areabnormally activated or disrupted by mutation in carcinogenesis have been identified.However, with the in-depth understanding of tumor, it is recognized that epigeneticmodifications play crucial roles in the initiation and development of human cancers inrecent years. Cancer is as much an epigenetic disease as it is a genetic disease, whichtogether lead to the malignant transformation of cells caused by the activation of oncogeneand the inactivation of tumor suppressor genes. Epigenetic alterations occurred even earlierthan the genetic mutations at a preneoplastic stage. As the smallest open reading frame andalso the most obvious overlap on the structure of the HBV genome region, HBX, encodesthe hepatitis B virus X protein (HBx) which has been implicated in HBV-related HCCpathogenesis and acts as a weak oncogene or a cofactor in hepatocarcinogenesis. Severalrecent studies have suggested that HBx is also involved in epigenetic regulation duringhepatocarcinogenesis.The overexpression of HBx can contribute to gene promoter hypermethylation and inturn to HCC by increasing total DNMT activity. However, the mechanism by which HBxactivates DNMTs expression remains unknown. MicroRNAs (miRNAs) are noncodingRNAs,19to25nucleotides long, which regulate gene expression by targeting mRNAsthrough base pairing at partially or fully complementary sites for cleavage or translationalrepression. They are involved in diverse biological functions including organismdevelopment, differentiation, proliferation, apoptosis, and stress response. Deviations fromnormal miRNA expression patterns play roles in human diseases, including cancers. SomemiRNAs may function as oncogenes or tumor suppressor genes (TSGs). Growing evidence supports a role for miRNAs as both targets and effectors in aberrant mechanisms of DNAhypermethylation.In this study, we investigated whether some miRNAs are aberrantly expressed andinvolved in the regulation of the abnormal DNA methylation status in HCC. We found thatthe expression of miR-152was down-regulated in the livers of HBx transgenic mice incomparison with the livers of wildtype mice by miRNA microarray and real-timepolymerase chain reaction in our previous studies. With in silico predictions, DNMT1wasdefined as a potential target of miR-152. Our results showed that down-regulated miR-152induced aberrant DNA hypermethylation by repressing expression of DNMT1inHBV-related HCC. The forced expression of miR-152in liver cell lines resulted in amarked reduction of the expression of DNMT1at both the mRNA and protein levels bydirectly targeting the3' untranslated regions of DNMT1. This in turn led to a decrease inglobal DNA methylation, whereas inhibition of miR-152caused global DNAhypermethylation and increased the methylation levels of two tumor suppressor genes,glutathione S-transferase pi1(GSTP1) and E-cadherin1(CDH1). And the expression ofmiR-152was frequently down-regulated in HBV-related HCC tissues in comparison withadjacent noncancerous hepatic tissues and was inversely correlated to DNMT1mRNAexpression in HBV-related HCCs.In this study, we characterized the epigenetic regulation roles of miR-152in HBV-related HCC; it may function as a tumor suppressor in HBV-HCC development, enrichesthe pathogenesis of HBV-HCC. Our results support a previously unreported role ofmiRNAs in the regulation of aberrant DNA methylation in HBV-related HCC. It plays apositive role for enriching the interaction regulating mechanism between differentepigenetic modifications, as well as for the understanding of the complexity of epigeneticregulatory networks. We also believe that these finding have potentially relevanttherapeutic implications. The results of this study provide a strong rationale for developingepigenetic therapies that use synthetic miR-152, alone or with other treatments, toreexpress the methylation-silenced TSGs and normalize aberrant patterns of methylation inHBV-related HCC. These findings support the potential development of miRNA-basedtargeted approaches for the treatment of HBV-related HCC.In addition to the small RNA molecules, another type of non-coding RNA (longnon-coding RNA, lncRNA) is gradually coming into view in recent years. LncRNAs are aclass of non-coding RNAs transcripts longer than200nucleotides, with little or no protein-coding capacity. Recent efforts have identified that lncRNAs also has a veryimportant role in epigenetic regulation. Regulating gene expression by a variety ofmechanisms, lncRNAs are involved in genomic imprinting, chromatin modification,transcription, post-transcriptional processing, protein function regulation and otherimportant signal transduction regulation. They are involved in diverse biological functions,and increasing evidence relates changes in expression levels of lncRNAs to complexdisease such as cancer. Then Whether HBV can affect the expression of lncRNAs topromote tumorigenesis?In the present study, we also investigated the expression alteration of lncRNAs byHBV, and the effects and molecular mechanisms of lncRNAs in HBV-HCC, start withinthe HBx transgenic mice perspective to eliminate other interfering factors. We screened anumber of lncRNAs that are significantly different expressed in the livers of HBxtransgenic mice and wild-type mice by lncRNAs microarray, and validated the expressionof these lncRNAs in expanded samples of transgenic and wild-type mice livers of differentage and sex by real-time quantitative PCR. Our results confirmed that HBx can really alterthe expression of lncRNAs; this indicated that HBV carriers may have its specificlncRNAs profile. And many of these lncRNAs are aberrantly expressed at a preneoplasticstage in early infection, suggesting that their expression differences may be closely relatedto the tumorigenesis. We found that HBx induced down-regulated lncRNA-AK050349caninhibit the proliferation and migration of tumor in vitro and in vivo, act as a tumor in thedevelopment of HBV-HCC. AK050349can combine with vimentin protein and inhibit itsexpression, and change the normal cytoskeleton structure to inhibitor the tumor metastasis.Our study characterized the important role of HBx-related lncRNAs in developmentof HCC; it is an important supplement to the HBV-HCC tumorigenesis and transfernetwork, allowing us to better understand the mechanisms of HCC, so as to get earlyfinding, early diagnose, and early prevention to improve the survival rate of HCC.Meanwhile, the tumor suppressor role of lncRNA-AK050349in the cascade reaction oftumor growth and metastasis in HBV-related HCC provides us new ideas and target for thetreatment of HBV-related HCC. The results of this study provide a strong rationale fordeveloping epigenetic therapies that use synthetic lncRNAs for the treatment ofHBV-related HCC.